The present invention relates to methods and compositions for the modulation of T-cell activity by GnRH-I and GnRH-II, and specific functional analogs of GnRH-I and GnRH-II receptors.
T-Cells in Immunity and Disease
Immune responses are largely mediated by a diverse collection of peripheral blood cells termed leukocytes. The leukocytes include lymphocytes, granulocytes and monocytes. Granulocytes are further subdivided into neutrophils, eosinophils and basophils. Lymphocytes are further subdivided into T and B lymphocytes. T-lymphocytes originate from lymphocytic-committed stem cells of the embryo. Differentiation occurs in the thymus and proceeds through prothymocyte, cortical thymocyte and medullary thymocyte intermediate stages, to produce various types of mature T-cells. These subtypes include CD4+ T cells (also known as T helper and T inducer cells), which, when activated, have the capacity to stimulate other immune system cell types. The T-helper cells are further subdivided into the Th1, Th2 and Th3 cells, primarily according to their specific cytokine secretion profile and function. T cells also include suppressor/regulator T cells (previously known as cytotoxic/suppressor T cells), which, when activated, have the capacity to lyse target cells and suppress CD4+ mediated effects.
T-cell activation: Immune system responses are elicited in a variety of situations. The most frequent response is as a desirable protection against infectious microorganisms. The current dogma is that in the organism, under physiological conditions, resting T-cells are activated and triggered to function primarily by antigens which bind to T-cell receptor (TCR) after being processed and presented by antigen-presenting cells, or by immunocyte-secreted factors such as chemokines and cytokines, operating through their own receptors. Experimentally, T-cells can be activated by various non-physiological agents such as phorbol esters, mitogens, ionomycin, and anti-CD3 antibodies. To identify novel physiological means directly activating and/or regulating T-cells in conditions of health and disease, especially in non-lymphoid environments (e.g. brain) and in a TCR-independent manner, remains a challenge of scientific and clinical importance.
In recent years, it has become evident that specific immune responses and diseases are associated with T-helper (Th) functions. Among these are anti-viral, anti-bacterial and anti-parasite immune responses, mucosal immune responses and systemic unresponsiveness (mucosally induced tolerance), autoimmune reactions and diseases, allergic responses, allograft rejection, graft-versus host disease and others. Furthermore, specific T-cell mediated proinflammatory functions may have either beneficial or detrimental effects on specific neoplasias: on the one hand, proinflammatory cytokines may assist in anti-tumor immune surveillance, and, on the other, elevated levels of proinflammatory cytokines were found within chronically inflamed tissues that show increased incidence of neoplasia.
In general, CD4+ T-cells can be divided into at least two major mutually exclusive subsets, Th1 and Th2, distinguished according to their cytokine secretion profile. Th1 cells secrete mainly INF-γ, TNF-β and IL-2, their principal effector function being in phagocyte-mediated defense against infections. The Th1 cells are usually associated with inflammation, and induce cell-mediated responses.
Essential and beneficial immunity cannot take place without Th1 cytokines, but their over or dis-regulated production leads to numerous detrimental clinical consequences. Th2 cells induce B-cell proliferation and differentiation, and thus, induce immunoglobulin production. Cytokines from Th2 cells (mainly IL-4, IL-10 and IL-13) can also antagonize the effects of Th1 cell-mediated reactivities, inhibiting potentially injurious Th1 responses.
T-cell migration and integrin-fibronectin binding: Adhesion is important for a cell: it provides anchorage, traction for migration, signals for homing and regulates growth and differentiation. In the immune system, the ongoing migration, extravasation and homing of T-cells from the blood stream to various tissues and organs is crucially dependent on various adhesive interactions with ligands on target cell-surfaces and matrices.
A class of glycoproteins has been identified as comprising the receptors in the cell recognition system for cell-extracellular matrix interaction. These proteins, referred to as integrins, are characterized by the involvement of the RGD sequence in ligand recognition, and appear to play a significant role in the assembly of the extracellular matrix (Ruoslahti, E. “Versatile Mechanisms of Cell Adhesion,” The Harvey Lectures, Series 84, pp 1-17 (1990)).
An integrin molecule is a heterodimeric membrane protein composed of one α and one β subunit. Several subunits of each kind are known, and various combinations of these subunits make up receptors with differing ligand specificities. The ligands for integrin are extracellular matrix proteins such fibronectin, lamanin, collagens and vitronectin or membrane proteins at the surface of other cells. By binding to their ligands, integrins mediate the adhesion of cells to extracellular matrices and to other cells.
Integrin functions have been shown to play a key role in a broad spectrum of normal and diseased conditions in general, and in inflammation and injury in particular. For example, T-cell recruitment into inflamed gingival tissues in periodontal disease (Taubman and Kawai, Crit. Rev Oral Biol Med 2001, 12(2) 125-35), and into the lamina propria in intestinal inflammation is associated with increased integrin expression. Normal cells are anchorage (integrin-fibronectin) dependent for progression through the cell cycle, whereas cancer cells exhibit anchorage-independent mitogenic activity. Furthermore, since resting T-cells cannot adhere, integrin-mediated fibronectin binding is indicative of significant activation and induction of T-cell function.
Three major events are involved in inflammation: (1) increased blood supply to the injured or infected area; (2) increased capillary permeability enabled by retraction of endothelial cells; and (3) migration of leukocytes out of the capillaries and into the surrounding tissue (Roitt et al., Immunology, Grower Medical Publishing, New York, 1989). Increased capillary permeability allows larger molecules to cross the endothelium that are not ordinarily capable of doing so, thereby allowing soluble mediators of immunity such as leukocytes to reach the injured or infected site. Leukocytes, primarily neutrophil polymorphs (also known as polymorphonuclear leukocytes, neutrophils or PMNS) and macrophages, migrate to the injured site by a process known as chemotaxis. At the site of inflammation, tissue damage and complement activation cause the release of chemotactic peptides such as C5a. Complement activation products are also responsible for causing degranulation of phagocytic cells, mast cells and basophils, smooth muscle contraction and increases in vascular permeability (Mulligan et al. 1991 J. Immunol. 148: 1479-1485).
The traversing of leukocytes from the bloodstream to extravascular sites of inflammation or immune reaction involves a complex but coordinated series of events. At the extravascular site of infection or tissue injury, signals are generated such as bacterial endotoxins, activated complement fragments or proinflammatory cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor (TNF) which activate leukocytes and/or endothelial cells and cause one or both of these cell types to become adhesive. Initially, cells become transiently adhesive (manifested by rolling) and later, such cells become firmly adhesive (manifested by sticking). Adherent leukocytes travel across the endothelial cell surface, diapedese between endothelial cells and migrate through the subendothelial matrix to the site of inflammation or immune reaction (Harlan et al., Adhesion-Its role in Inflammatory Disease, W. H. Freeman & Co., New York, 1992).
Although leukocyte traversal of vessel walls to extravascular tissue is necessary for host defense against foreign antigens and organisms, leukocyte-endothelial interactions often have deleterious consequences for the host. For example, during the process of adherence and transendothelial migration, leukocytes release oxidants, proteases and cytokines that directly damage endothelium or cause endothelial dysfunction. Once at the extravascular site, emigrated leukocytes further contribute to tissue damage by releasing a variety of inflammatory mediators. Moreover, single leukocytes sticking within the capillary lumen or aggregation of leukocytes within larger vessels are responsible for microvascular occlusion and ischemia. Leukocyte-mediated vascular and tissue injury has been implicated in pathogenesis of a wide variety of clinical disorders such as acute and chronic allograft rejection, vasculitis, rheumatoid and other forms of inflammatory based arthritis, inflammatory skin diseases, adult respiratory distress syndrome, ischemia-reperfusion syndromes such as myocardial infarction, shock, stroke, organ transplantation, crush injury and limb replantation.
Many other serious clinical conditions involve underlying inflammatory processes in humans. For example, multiple sclerosis (MS) is an inflammatory disease of the central nervous system. In MS, circulating leukocytes infiltrate inflamed brain endothelium and damage myelin, with resultant impaired nerve conduction and paralysis (Yednock et al., 1992 Nature 366: 63-66). Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of tissue damage caused by self antigen directed antibodies. Auto-antibodies bound to antigens in various organs lead to complement-mediated and inflammatory cell mediated tissue damage (Theofilopoubs, A. N. 1992 Encyclopedia of Immunology, pp. 1414-1417).
Reperfusion injury is another condition associated with activation of the inflammatory system and enhanced leukocyte-endothelial cell (EC) adhesion. There is much evidence that adhesion-promoting molecules facilitate interactions between leukocytes and endothelial cells and play important roles in acute inflammatory reaction and accompanying tissue injury. For example, in acute lung injury caused by deposition of IgG immune complexes or after bolus i.v. infusion of cobra venom factor (CVF), neutrophil activation and the generation of toxic oxygen metabolites cause acute injury (Mulligan et al., 1992 J. Immunol. 150(6): 2401-2405). Neutrophils (PMNs) are also known to mediate ischemia/reperfusion injury in skeletal and cardiac muscle, kidney and other tissues (Pemberton et al., 1993 J. Immunol. 150: 5104-5113). Infiltration of airways by inflammatory cells, particularly eosinophils, neutrophils and T lymphocytes are characteristic features of atopic or allergic asthma (Cotran et al., Pathological Basis of Disease, W. B. Saunders, Philadelphia, 1994). Cellular infiltration of the pancreas with resultant destruction of islet beta-cells is the underlying pathogenesis associated with insulin-dependent diabetes mellitus (Burkly et al. 1994 Diabetes 43: 529-534).
Activation of inflammatory cells whose products cause tissue injury underlies the pathology of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Neutrophils, eosinophils, mast cells, lymphocytes and macrophages contribute to the inflammatory response. Minute microabcesses of neutrophils in the upper epithelial layers of the dermis accompany the characteristic epidermal hyperplasia/thickening and scaling in psoriasis.
Various anti-inflammatory drugs are currently available for use in treating conditions involving underlying inflammatory processes. Their effectiveness however, is widely variable and there remains a significant clinical unmet need. This is especially true in the aforementioned diseases where available therapy is either of limited effectiveness or is accompanied by unwanted side effect profiles. Moreover, few clinical agents are available which directly inhibit cellular infiltration, a major underlying cause of tissue damage associated with inflammation. Thus, there is a need for a safe, effective clinical agent for preventing and ameliorating cellular infiltration and consequential pathologic conditions associated with inflammatory diseases and injuries.
Modification of T-cell activity: Therapeutic application of T-cell modulating agents has been proposed for the treatment of conditions characterized by both immune deficiency and chronic inflammation. For example, U.S. Pat. No. 5,632,983 to Hadden discloses a composition consisting of peptides of thymus extract, and natural cytokines, for stimulation of cell mediated immunity in immune deficient conditions. Although significant enhancement of a number of cell mediated immune functions was demonstrated the effects were highly non-specific, as could be expected when employing poorly defined biologically derived materials.
Recently, Butcher et al. (U.S. Pat. No. 6,245,332) demonstrated the specific interaction of chemokine ligands TARC and MDC with the CCR4 receptors of memory T-cells, enhancing interaction of these cells with vascular epithelium and promoting T-cell extravasation. Therapeutic application of CCR4 agonists was disclosed for enhanced T-cell localization, and of antagonists for inhibition of immune reactivity, as an anti-inflammatory agent. Although the ligands were characterized, and identified in inflamed tissue, no actual therapeutic effects of agonists or antagonists were demonstrated.
Inhibition of a number of T-cell functions has been the target of many proposed anti-inflammatory therapies. Haynes et al. (U.S. Pat. No. 5,863,540) disclosed the use of anti-CD44 (cell adhesion molecule effecting T-cell activation) antibody for treatment of autoimmune conditions such as Rheumatoid Arthritis. Godfrey et al. (U.S. Pat. No. 6,277,962) disclosed a purified ACT-4 T-cell surface receptor expressed in activated CD4+ and CD8+ T-cells, and proposed the use of anti-ACT-4 antibodies to achieve downregulation of T-cell activation. Similarly, Weiner et al. (U.S. Pat. Nos. 6,077,509 and 6,036,457) proposed treatment with peptides containing immunodominant epitopes of myelin basic protein (associated with Multiple Sclerosis) for the specific suppression of CD4+ T-cell activity in this central nervous system autoimmune condition. However, none of the proposed applications were able to demonstrate any specific effect on the processes regulating expression of T-cell specific surface proteins responsible for immune activity.
Autoimmune Diseases
Autoimmune diseases are characterized by the development of an immune reaction to self components. Normally, tissues of the body are protected from attack by the immune system; in autoimmune diseases there is a breakdown of the self-protection mechanisms and an immune response directed to various components of the body ensues. Autoimmune diseases are for the most part chronic and require life long therapy. The number of recognized autoimmune diseases is large and consists of a continuum ranging from diseases affecting a single organ system to those affecting several organ systems. With increased understanding of the molecular basis of disease processes, many more diseases will likely be found to have an autoimmune component. Autoimmune diseases are typically divided into Organ Specific, and Non-Organ Specific Autoimmune disease. Specific examples of Organ Specific Autoimmune diseases are: Hashimoto's thyroiditis, Graves' disease, Addison's disease, Juvenile diabetes (Type I), Myasthenia gravis, pemphigus vulgaris, sympathetic opthalmia, Multiple Sclerosis, autoimmunehemolytic anemia, active chronic hepatitis, and Rheumatoid arthritis.
Rheumatoid arthritis is a systemic, chronic, inflammatory disease that affects principally the joints and sometimes many other organs and tissues throughout the body, characterized by a nonsuppurative proliferative synovitis, which in time leads to the destruction of articular cartilage and progressive disabling arthritis. The disease is caused by persistent and self-perpetuating inflammation resulting from immunologic processes taking place in the joints. Both humoral and T-cell mediated immune responses are involved in the pathogenesis of rheumatoid arthritis.
The key event in the pathogenesis of the arthritis is the formation of antibodies directed against other self antibodies. T cells may also be involved in the pathogenesis of rheumatoid arthritis. A large number of T cells are found in the synovial membrane, outnumbering B cells and plasma cells. Additionally, procedures to decrease the population of T cells (such as draining the thoracic duct) result in remission of symptoms.
Rheumatoid arthritis is a very common disease and is variously reported (depending on diagnostic criteria) to affect 0.5 to 3.8% of women and 0.1 to 1.3% of men in the United States.
Multiple sclerosis is a neurogenic disease that is thought to be caused by autoimmune mechanisms. The systemic immune response and the response of the central nervous system become involved. Although the cause and pathogenesis of multiple sclerosis are unknown, it is widely believed that immune abnormalities are somehow related to the disease. Suppression or modulation of the immune responses may be the key. Multiple sclerosis is modeled, in rodents, by the passive transfer of immune reactivity to Myelin Basic Protein via administration of sensitized T-cell (experimental autoimmune encephalomyelitis: EAE).
Myasthenia gravis is another nervous system related autoimmune disorder caused by antibodies directed against the acetylcholine receptor of skeletal muscle. In both experimental allergic myasthenia gravis and human myasthenia gravis, the extent of acetylcholine receptor loss parallels the clinical severity of the disease, suggesting that acetylcholine receptor antibody-induced acceleration of acetylcholine receptor degradation is important in the development of myasthenia gravis.
Other disorders, especially those presumed to be autoimmune in origin, can occur in association with myasthenia gravis. Thyroid disease, rheumatoid arthritis, systemic lupus erythematosus, and pernicious anemia all occur more commonly with myasthenia gravis than would be expected by chance.
One example of a non-organ specific Autoimmune disease is Systemic lupus erythematosus.
Acute attacks of Systemic lupus erythematosus are usually treated by adrenocortical steroids or immunosuppressive drugs. These drugs often control the acute manifestations. With cessation of therapy the disease usually reexacerbates. The prognosis has improved in the recent past; approximately 70 to 80% of patients are alive 5 years after the onset of illness and 60% at 10 years. Lifelong therapy is required to control the disease.
The foundation of therapy of autoimmune diseases is treatment with immunosuppressive agents. The basis for this therapy is attenuation of the self-directed immune response with the primary aim being to control symptoms of the particular disease. The drugs utilized to achieve this aim are far from satisfactory, in that adverse side effects are numerous and control of the disease is many times difficult to achieve. The problem is compounded by the chronicity of the disease with effective therapy becoming more difficult with time. An indication of the severity of particular diseases is seen in the willingness to accept greater risks associated with therapy as the disease progresses. Currently available therapy is distinctly non-selective in nature, having broad effects on both the humoral and cell mediated arms of the immune system. This lack of specificity can limit the effectiveness of certain therapeutic regimens. The main groups of chemical immunosuppressives are alkylating agents, antimetabolites, corticosteroids, and antibiotics, each will be discussed briefly.
The corticosteroids, also called adrenocorticosteroids, are fat-like compounds produced by the outer layer or cortex, of the adrenal gland. Therapeutic use of the corticosteroids for autoimmune disease is based on their two primary effects on the immune system, anti-inflammatory action and destruction of susceptible lymphocytes. They also effect a redistribution of lymphocytes from peripheral blood back to the bone marrow. The use of corticosteroids is not without adverse side effects however, particularly during the course of life-long treatment which is required for many of the autoimmune diseases.
Major side effects of steroids are: Cushing syndrome, muscle atrophy, osteoporosis, steroid induced diabetes, atrophy of the adrenal glands, interference with growth, susceptibility to infections, aseptic bone necrosis, cataract development, gastric ulcer, steroid psychosis, skin alterations and nervous state accompanied by insomnia.
Attempts to minimize side effects incorporate alternate day or less frequent dosage regimens.
Another recently developed immunosuppressive agent is the antibiotic cyclosporin A. The antibiotic has greatest activity against T cells and does not seem to have much direct effect on B cells. The drug is being evaluated for the treatment of autoimmune diseases for which it shows some promise. Side effects include hair growth, mild water retention, renal toxicity, and, in older patients, nervous system disorders symptoms have been observed.
Other drugs are used alone or in combination with those listed above and include gold salts and antimalarials, such as chloroquine. Another class of drugs, the non-steroidal anti-inflammatory drugs are used extensively in arthritis. These drugs provide analgesia at low doses and are anti-inflammatory after repeated administration of high doses. Nonsteroidal anti-inflammatory drugs all act rapidly and their clinical effects decline promptly after cessation of therapy. However, they do not prevent the progression of rheumatoid arthritis, do not induce remissions, and are frequently associated with dangerous gastrointestinal side effects. Immunostimulants, such as levamisol have also been used in many autoimmune diseases but side effects have generally limited their use. Clearly, new therapies and drugs for the treatment of autoimmune disorders are needed.
Lymphocytes in Cancer Immunotherapy
Immunotherapy in cancer patients is usually directed to the production or stimulation of populations of reactive antitumor lymphocytes, to provide specific and natural cytotoxic effects directed against highly expressed tumor antigens. Many studies have reported successful immunization of human and non-human subjects with cancer antigens to stimulate circulating cytotoxic T-cell precursors (see, for example, Rosenberg, S A et al, Nature Med 1998; 4: 321), however, this has not yet correlated with any clinically significant effect. In another method for stimulation of anti-tumor immunotoxicity, Ostrand-Rosenberg et al (U.S. Pat. No. 6,319,709) disclosed the ex-vivo modification of tumor cells, and their re-introduction into the patient, to stimulate a beneficial anti-cancer immune response.
Another approach is the adoptive transfer of selected and expanded sub-populations of anti-tumor lymphocytes. For example, adoptive transfer of tumor infiltrating lymphocytes (TIL), along with interleukin-2 treatment, can mediate the regression of established lung and liver metastases (Rosenberg, S. A., et al., Science 1998; 233: 1318-1321). However, engraftment and persistence of the transferred cells has not been generally observed. Recent reports of successful tumor regression in melanoma patients receiving clonal repopulation with antitumor lymphocytes, following lymphodepletion, also emphasized the requirement for IL-2 treatment, and the danger of autoimmune side-effects (Dudley, M E et al, Science 2002; 298: 850-54). Thus there is a great need for new anti-cancer T-cell therapies.
Neurotransmitters and Immune System Function
It is generally accepted that the immune, nervous and endocrine systems are functionally interconnected. The significance of direct neuronal signaling on immune system components, including T-cells, can be appreciated considering the extensive innervation of all primary and secondary lymphoid tissue; the presence of both peptidergic and non-peptidergic neurotransmitters in capillaries and at sites of inflammation, injury or infection; and the demonstrated expression of specific receptors for various neurotransmitters on T-cell (and other immune system components) surface membrane.
Specific modulation of immune function has been demonstrated for a number of neurotransmitters. Recently, neuropeptides somatostatin (SOM), calcitonin gene related peptide (cGRP), neuropeptide Y (NPY) and also Dopamine were found to interact directly with specific receptors on the T-cell surface, while substance P (Sub P) indirectly affected T-cell function. These neurotransmitters exert both inhibitory and stimulatory influence on T-cell cytokine secretion, adhesion and apoptosis, depending on T-cell lineage and activation states (Levite, M.: Nerve Driven Immunity. The direct effects of neurotransmitters on T-cell function. Ann NY Acad. Sci. 2001 917: 307-21). Similarly, physiological concentrations of the neurotransmitters SOM, Sub P, cGRP and NPY were found to directly induce both typical and non-typical cytokine and chemokine secretion from T-cells and intestinal epithelium, thus either blocking or evoking immune function (Levite, M. Nervous immunity: neurotransmitters, extracellular K+ and T-cell function. Trends Immunol. 2001 January; 22(1): 2-5). Clearly, immune function is sensitive to neurogenic control.
A number of therapeutic applications of immune modulation by manipulation of neurotransmitters have been proposed. In one, botulinum toxin's peptide-lytic activity is employed to reduce the effect of immune-active neurotransmitters Sub P, cGRP, NK-1, VIP, IL-1 and IL-6 and others on neurogenic inflammatory conditions such as arthritis, synovitis, migraine and asthma (U.S. Pat. No. 6,063,763 to First). Hitzig (U.S. Pat. No. 5,658,955) proposes the combined application of neurotransmitters Dopamine and serotonin for complex inhibition and stimulation of various immune functions, for the treatment of AIDS and HIV infection, cancers, migraine, autoimmune inflammatory and allergic conditions, chronic fatigue syndrome and fibromyalgia. On the whole, however, the immune modulation of these inventions is of a broad and non-specific nature, with significant likelihood of undesirable complications and side effects in practice. In addition, no clear mechanism of action was defined for the immune-modulatory effects of Dopamine and serotonin in the latter disclosure. Thus, there is a need for improved methods of modulation of immune function via specific neuropeptides and defined pathways of immune activation.
GnRH-I and GnRH-II: The Gonadotropin releasing hormone-II (GnRH-II), is a unique ten amino acid long neuropeptide, which is conserved throughout 500 million years of evolution, and has recently been identified in the brain and non-neural (kidney, bone marrow, prostate and placenta) tissues of various mammals. The peptide structure of GnRH-II shares 70% homology with that of the known mammalian neurohormone, GnRH (GnRH-I), but is encoded by its own gene. In contrast to GnRH-II, which is the prime regulator of reproduction, GnRH-II exerts only very mild effects on reproduction in mammals, and its principal physiological role remains unclear (see, for example, Fink, G. Gonadotropin secretion and its control; in The Physiology of Reproduction (eds Knobil, E. & Neill, J. D.) 1349-1377 (Raven Press, New York, 1988). The two GnRH isoforms are produced mainly in areas of the brain stem and hypothalamus, with axons of the hypothalamic GnRH neurons terminating in the infundibulum, close to the fenestrated portal capillary plexus. The strict evolutionary conservation of GnRH-II, from primitive vertebrates to mammals, taken together with its different functional profile as compared to GnRH-I, suggests that it may have different, yet undiscovered, important physiological functions.
Surprisingly, it was recently demonstrated that the promoters of GnRH-I and GnRH-II are differentially regulated, suggesting distinct physiological functions for the two isoforms (Chen et al. Transcriptional regulation of the human gonadotropin-releasing hormone II gene is mediated by a putative cAMP response element. Endocrinology, In Press August, 2001). In bullfrogs, GnRH-II, and GnRH-I, to a lesser extent, have sympathetic neurotransmitter function. Although only one GnRH receptor has been characterized in mammals, high affinity receptors binding GnRH II have been demonstrated in catfish and goldfish, suggesting that additional GnRH II receptors may also be present in mammals. Niell (Neill J D et al, Arch. Physiol Biochem 2002; 110: 129-36) described a putative human GnRH-II receptor mRNA (NCBI Accession number NM 057163), sharing only 55% homology with the human GnRH-II receptor, identified on the basis of sequence homology with non-human species GnRH-I receptor. However, the function and identity of this putative receptor have yet to be definitively determined.
Analogs of GnRH are commonly used for intervention in the reproductive cycles and behavior of mammals and lower vertebrates (see, for example, Millar, R P et al. J. Biol. Chem. 1989, 264: 21007-013). Thus, certain modifications in GnRH structure (for example, positions 8, 9 and 10) have been recognized to confer characteristically agonist properties, while others (positions 1, 2, 3 and 6) produce antagonist analogs. These effects seem to demonstrate species, and growth-stage specific variance. In clinical application, native GnRH peptides have demonstrated only minimal potency via oral administration, and pharmaceutical compositions of GnRH analogs have been proposed, for example, for modulation of sex hormone levels in mammals (U.S. Pat. No. 5,140,009 to Haviv et al.) and treatment of male pattern baldness (U.S. Pat. No. 5,574,011 to Tein).
Walsh et al. and Goulet et al. (U.S. Pat. Nos. 6,228,867 and 5,985,901, respectively) disclose the application of a variety of non-peptide GnRH antagonists for the treatment of endometriosis, uterine fibroids, prostate, ovarian and mammary cancer, PMS, irritable bowel syndrome, precocious puberty, and for use in contraception and assisted fertilization techniques. The non-peptide analogs are emphasized for their superior oral potency, and all of the disclosed embodiments are based on the inhibition of Leutinizing Hormone and Follicle Stimulating Hormone release.
Analogs of GnRH are known to inhibit the growth of gonadal steroid-dependent tumors by both sex hormone deprivation and a direct effect on the cancer cells. Thus, GnRH has been applied, for example, for preoperative androgen block in prostate adenocarcinoma (Sharkey, J. et al. J. Endourol 2000 May; 14(4): 343-50) and in combination with tamoxifen in treatment of breast cancer (Klijn, J G et al., J. Clin Oncol 2001 Jan. 15; 19(2) 343-53). Direct effects are presently unclear, however: although many mammary cancer cells, for example, express GnRH binding sites, some also express the GnRH gene, suggesting autocrine and/or paracrine effects in these cells. Effective inhibition of mammary tumor growth is achieved with relatively high dosage of GnRH and its analogs. Attempting to avoid some of the disadvantages of GnRH I and GnRH II analogs, Lovas et al. (U.S. Pat. No. 5,593,965) has disclosed the therapeutic use of Lamprey GnRH-II, a natural GnRH analog lacking mammalian gonadotropic activity, for inhibiting mammary tumor proliferation. No mention is made of neuroimmune interaction, inhibition of steroid independent cancers or ex-vivo treatment and re-introduction of autologous immune cells.
Non-gonadal cancers may also express GnRH binding sites: colon adenocarcinoma cells were effectively inhibited in vitro and in vivo by exposure to the chimeric protein L-GnRH-PE66 (Ben-Yehudah, A. et al. Int J Cancer 2001 Apr. 15; 92(2): 263-8), targeting the cancer cells with the GnRH peptides. However, toxicity, associated pain and the need for frequent administration were reported disadvantages of the treatment.
Alterations in immune function have been observed in correlation with administration of GnRH or analogs, in both clinical and experimental studies. For example, GnRh antagonist treatment of neonatal rhesus monkeys and marmosets resulted in reduced T-cell proliferation and impaired resistance to disease (Mann, D R et al Am J Reprod Immunol 2000; 44: 30-40), while women receiving GnRH agonists for endometriosis demonstrated increased T-cell and NK cell counts (Hsu C C et al Obstet Gynecol 1977; 89: 993-8). However, these effects involved complex endocrine and metabolic interactions, and no demonstration of direct effects of GnRH of T-cells were observed.
Neuroprotective Immunity: In the context of neuroimmune interaction, the recent discovery of neuroprotective interactions between T-cells and neuronal tissue in neurotoxicity, disease and injury is intriguing. Several studies by Schwartz, et al have shown that T-cell deficient mice are more susceptible to experimentally induced neuronal injury and neurotoxicity, and that reconstitution with wild-type splenocytes can effectively restore resistance. Additional evidence for such protective autoimmunity in CNS trauma was provided by the demonstration of potentiation of neuronal survival by prior, unrelated CNS insult in autoimmune encephalomyelitis-resistant strains of mice (see, for example, Yoles, et al, J Neurosci 2001, Jun. 1; 21(11): 3740-48; Kipnis, et al, J Neurosci 2001 Jul. 1; 21(13): 4564-71; and Schori, et al, J Neuroimmunol 2001 Oct. 1: 119(2): 199-204). Clinical application of such neuroprotective immunity has been proposed, employing vaccination with non-pathogenic CNS derived peptides such as MBP to boost innate beneficial autoimmunity (Schwartz and Kipnis, Trends Mol Med 2001 June; 7(6): 252-58; and Schwartz, Surv Ophthalmol 2001 May; 45 Suppl 3: S256-60) and stimulation of peripheral monocytes for enhancement of axonal regeneration (U.S. Pat. No. 6,117,242 to Eisenbach-Schwartz). No mention is made of GnRH or GnRH analog modulation of T-cell activity, and furthermore, the authors note the substantial risk of inducing undesired autoimmune disease using immunization with self antigens.
Studies of lymphocyte activation in other neurogenic conditions also indicate a potential neuroprotective role of immune cells: in patients with encephalitis and MS, the beneficial brain-derived-neurotrophic-factor BNDF is secreted by immune cells in response to CNS auto-antigen stimulation (Kerschensteiner, et al, J Exp Med 1999 Mar. 1; 189(5): 865-70). Furthermore, in clinical trials of an altered peptide ligand of myelin basic protein administered to patients with relapsing-remitting MS, reduction in lesion volume and number was achieved in the MBP-treated patients compared to the placebo group. However, the dosage required was high (5 mg), and the trial was suspended due to undesirable side effects (hypersensitivity). No mention was made of GnRH stimulation of T-cells.
Neuroimmunology and Psychopathology: Many studies have demonstrated significant correlation between immune function and a variety of emotional and psychopathological conditions, especially schizophrenia and suicide (see, for example, Sperner-Unterweger B, et al, Scizophr Res 1999; 38: 61-70; Staurenghi A H, et al Psychoneuroendocrinology 1997; 22: 575-90; van Gent T, et al J Child Psychol Psychiatry 1997; 38: 33749; Nassberger L and Traskman-Bendz L Acta Psychiatr Scand 1993; 88: 48-52; and Dabkowska M and Rybakowski J Psychiatr Pol 1994; 28: 23-32). Presently it remains unclear whether the dysfunctional immune responses observed contributeo the psychopathogenic processes, are secondary to them, or a combination of the two.
T-cell enhancement has been observed in schizophrenia, and has been suggested as a marker of therapeutic outcome or neuroleptic treatment (Muller, et al Acta Psychiatr Scand 1993; 87: 66-71 and Sperner-Unterweger B et al Scizophr Res 1999; 38: 61-70). The authors made no mention of T-cell-related therapy or GnRH modulation of T-cell activity for treatment or prevention of the abovementioned disorders.
Manipulation of immune cells for therapy of brain related disorders has been proposed by Wank (Intern Pats. WO9950393A2 and WO9950393A3 to Wank, R). Wank describes the in-vitro activation of peripheral blood monocytes (PBMC), or phagocytes, for the treatment of a variety of brain-related disorders, including psychoses, schizophrenia, autism, Down's syndrome, disturbances of cerebral development and brain injury, based on the observation of inadequate immune responses in these conditions. In a report documenting adoptive immunotherapy of patients suffering from bipolar disorder, schizophrenia or autism, Wank describes the in-vitro activation, and reintroduction of the patients' own T-cells, in order to combat “chronically infected”, understimulated lymphocytes thought associated with these disorders. In this form of therapy, the T-cells are not stimulated directly, rather via monoclonal antibodies against the CD3 polypeptide complex, and IL-2. The patients were required to endure numerous weekly treatments (up to 104 weeks in one patient), and although improvement in some symptoms was noted, additional therapies were continued during and after these trials of adoptive immunotherapy. No mention is made of direct stimulation of T-cells with neuropeptides, of specific T-cell response to therapy, or of treatment with GnRH or GnRH analogs.
To date, the dynamics of GnRH interaction with specific GnRH receptors on normal and diseased human T-cells have not been addressed directly.
While reducing the present invention to practice, the present inventor has uncovered, for the first time, that physiological concentrations of GnRH, acting directly on-T cells via well characterized GnRH receptors, can modify numerous important T cell functions, such as, for example, induction of gene expression, most significantly of the 67 kD non-integrin laminin receptor, adhesion to laminin, chemotaxis and T-cell extravasation. Whereas GnRH effects on T cells have been previously unknown, the present invention surprisingly demonstrates that GnRH I and GnRH II act directly to modulate specific gene expression, and upregulate GnRH expression and secretion in normal and cancerous human T cells. Thus, the present invention provides methods for the modulation of T-cell activity by GnRH and specific GnRH receptor functional analogs and, more particularly, methods for the treatment of bacterial, viral, fungal infectious and parasitic diseases, containment of auto-immune and other injurious inflammatory processes, inhibition and prevention of tumor growth and dissemination, and prevention of host rejection of engrafted tissue employing GnRH receptor-mediated regulation of T-cell laminin-binding activity and extravasation, devoid of the above limitations.